Background: Bifidobacteria levels in saliva were found to be significantly correlated in adults with dental caries but less information available in the literature regarding its role in children. Aim: The aim is to compare the salivary levels of Bifidobacteria in children who are caries free with that of early childhood caries (ECC) and severe ECC (S-ECC). Materials and Methods: Saliva was collected using the tongue-loop method from a total of 60 children between the age group of 3–5 years and they were further divided into 3 groups. In addition, the age and gender of the children, sugar amount in diet, sugar frequency in diet, were recorded. Results: Bifidobacteria was isolated from all the three groups, but more were from S-ECC, followed by ECC and very few cases of caries-free children and was found to statistically significant (P < 0.001). Salivary levels of Bifidobacteria were significantly correlated with amount of sugar in the diet and frequency of sugar consumption. Conclusions: Salivary levels of Bifidobacteria were significantly associated with S-ECC and ECC, followed by caries free group. In future, it can be used as a salivary marker for caries risk assesment.

Bifidobacterium is classified as Gram-positive, nonmotile, often branched anaerobic bacteria. They are more commonly found in gastrointestinal tract, vagina some species also found in mouth but in low prevalence. Bifidobacteria and related genera, Scardovia, and Parascardovia, has been detected in the oral cavity but not always, in relation to active caries.[1] Various methods have been used to isolate or identify the presence of these bacteria in oral samples and has a significant influence on the detection rates. Modesto et al.[1] have reported the presence of Bifidobacteria and related genera in 13 of 19 samples of infected dentine and in 11 of 15 supragingival plaque samples. The selective medium used for the isolation of Bifidobacteria and related genera is mupirocin and a low pH environment. Using this approach, Bifidobacteria was isolated from the saliva of 94.2% of 192 adults[2],[3] at concentrations not significantly different to those of mutans streptococci but significantly greater than those of lactobacilli with Bifidobacterium dentium most frequently isolated species. High levels of Bifidobacteria were found in active root carious lesions compared to inactive root caries lesions in adults.[4] Bifidobacteria were also detected in occlusal carious lesions in both adults and children.

Saliva has been used as a diagnostic tool in caries with salivary levels of mutans streptococci, lactobacilli or yeasts being used individually or together to determine current caries activity. In a multivariate analysis, it has been found that salivary level of Bifidobacteria was more significantly associated with caries experience in adults than were the levels of mutans streptococci, lactobacilli, or yeasts.[5] Information regarding its role in children is still in question Therefore, this study was planned to compare the salivary levels of Bifidobacteria in children who are caries free with that of early childhood caries (ECC) and severe ECC (S-ECC).

Materials and Methods

Subjects

Sixty children between the age group of 3–5 years having primary dentition reporting to Department of Pedodontics and preventive dentistry Vinayaka Missions dental college Salem were randomly included in this study. They were further divided into Group 1 caries free children, Group 2 ECC, and Group 3 S-ECC. Children taking antibiotics or completed a course of antibiotics within the past 4 weeks, patients not willing to participate, with no primary teeth, with tooth structure defects, undergoing orthodontic treatment, or wearing a removable appliance were excluded from the study. Ethical approval for collection of saliva samples was obtained and written informed consent was obtained from parents.

Saliva sample processing

Saliva sample was collected using a 10-uL bacteriological Loop, which was drawn down the center of the anterior two-thirds of the tongue of each child until the loop was full of saliva equivalent to 10 uL of saliva.[6] The saliva sample was immediately dislodged into 1 ml Fastidious Anaerobic Broth, effecting a dilution of 1 in 100 of the saliva sample. The samples were placed in ice box and processed within 1–3 h. Duplicate 100 uL samples were spread onto the Mupirocin-containing selective medium[3] which is used for the isolation of Bifidobacteria. The difference in the salivary Bifidobacterial counts of the three groups was expressed as log10 (CFU/mL of saliva).

Mean values of microbial counts were compared using ANOVA test. Spearman correlation coefficients and Chi-square test were used to calculate relation between variables and salivary levels of microbial counts. All analyses were performed using SPSS software (version 19, SPSS Inc., IL., Chicago, USA).

Results

The mean age in this study was found to be between 4 and 4.5 years. Mean value of bifidobacteria level in caries free group was found to be 6.05, ECC group was found to be 19.22, and S-ECC group was 23.83 this shows that salivary levels of Bifidobacteria was found to be higher in S-ECC followed by ECC and caries free group as shown in [Table 1], the result was found to be statistically significant P < 0.001. However, children with S-ECC was found to be consuming high amount of sugar in diet and the levels of Bifidobacteria and the amount of sugar in diet showed a positive correlation and the result were statistically significant (P < 0.001) [Table 2]. Frequency of sugar intake was found to be more in children with S-ECC, and the levels of Bifidobacteria and the frequency of sugar intake showed positive correlation and the result were statistically significant (P < 0.001) [Table 3].

It has been proven that cariogenic bacteria involved in ECC are Streptococcus mutans and lactobacillus,[8],[9],[10],[11] but there are studies which proves the existence of other bacterial species as well. Bifidobacteria and its related species which has the ability to with stand in acidic environment, also known as acidogenic species which are mainly isolated from gut, but many studies proves their existence in oral cavity as well, but their presence was found to be very low. In this study, we have not identified isolates of Bifidobacteria but we have used the same analytical methods similar to the study reported by Modesto et al.[1] Mantzourani et al. in 2009 reported that the amount of Bifidobacteria isolated from occlusal carious lessions in children and adult was found to be equivalent to streptococcus mutans[11],[12],[13],[14] in our study the levels of Bifidobacteria was found to be higher in S-ECC followed by ECC and a very low count in caries free children which is similar to the study reported by Kaur et al. in 2013 in which high count of Bifidobacteria was found in caries active children.[15],[16] Mantzourani et al. in 2010 reported that how the bifidobacteria colonize inside the oral cavity is still unknown[17] but it is believed that it is more commonly found on the open dentinal lesions that are caries free surface, similar to the observation of our study. The salivary levels of Bifidobacteria were significantly co-related with amount of sugar in diet and frequency of sugar intake[15] both these factors were found to be more in S-ECC which is similar to the study reported by Kaur et al. in 2013. However, role of Bifidobacteria in the caries progression, its adaptation to the oral cavity and isolation of strains of Bifidobacteria requires further investigation.

Conclusions

The salivary levels of Bifidobacteria was found to be higher in S-ECC group followed by ECC and caries free group

In future more investigations need to be carried out to determine the role of bifidobacteria in caries process, and also to identify their characteristics which enables them for colonization in oral cavity. Hence, the salivary levels of Bifidobacteria may serve as a marker of caries risk.

Acknowledgment

This study was supported by Department of Pedodontics and Preventive Dentistry Vinayaka mission's dental college Salem in association with Department of Microbiology Vinayaka mission's Kirupananda Variyar Medical College.